The long-term goal of this project is to understand the mechanisms by which ion channels in the vestibular hair cell membrane modulate the cell's membrane potential. This information is important because the magnitude of the membrane potential determines, among other things, the cell's sensitivity and tuning properties. In a first set of experiments, (specific aim 1), 4 different chimeras will be inserted into the extracellular loop (ECL) motif of the inward rectifier ion channel protein pKir2.1. This channel is found in the majority of native avion vestibular hair cells. The effects of the chimera insertion will be assayed by making single channel recordings from the mutated molecules expressed in CHO cells. It will be determined if changes in amino acids in the ECL change the unusually high open probability, P(O) seen in unmutated pKir2.1 during large hyperpolarizations. There are 2 sub aims of this first set of experiments. The first is to determine which and what is the smallest number of amino acids that have to be mutated to significantly change the shape of the P(O) function during hyperpolarizing voltages. The second is to measure single channel responses from native hair cells and compare them to those obtained from expressing the unmutated pKir2.1 channel in CHO cells. The intracellular environments of the two systems are different and that may influence the signaling pathways and in turn P(O). The second set of experiments (specific aim 2) concern the modulation of pKir2.1 by muscarinic receptor activation. Pilot data is presented showing that application of a muscarinic agonist, carbachol, to a cloned M3 receptor subtype expressed in a tSA-201 cell inhibits pKir2.1 currents in that cell; thereby depolarizing the cell's membrane potential. The probable pathway involved in this result is: the muscarinic M3 receptor subtype as the ligand receptor, coupled by a G protein with a particular alpha subunit (G-alpha-q/11) coupled to a particular K+ channel effector, pKir2.1. Stimulation of the cholinergic efferent vestibular system causes excitation of most primate vestibular primary afferents. The pathway by which presynaptic structures such as hair cells are depolarized, causing excitation of primary afferents, is unknown. We hypothesize that the pathway cited above is a most promising candidate. We propose to insert expression DNA for M3, and pKir2.1 from pigeon vestibular epithelia into tsA-201 cells, which endogenously express G-alpha-q/11, then demonstrate, using patch clamp assays, that M3 activation blocks the pKir2.1 channel causing a depolarization of the cell's membrane potential (sub aim 1). Next, we will clone G-alpha-q/11 and M5 (sub aim 2) and stably transfect M3/M5, G-alpha-q/11 and pKir2.1 into CHO cells and assay macroscopic currents and membrane voltages using using patch clamp techniques. Finally, we will apply M3 or M5 agonists and measure the macroscopic pKir2.1 currents in native cells in an epithelial slice (sub aim 3). The two specific aims of this project are linked in that one drives studies of the the effector pKir2.1 and the second directs studies the msucarinic receptors in the pathway proposed above.